The invention relates to aqueous L-lysine-containing animal feed supplements, which optionally inactivated microorganisms from the fermentation process, and a process for the production of such supplements.
The essential amino acid L-lysine is widely used as an animal feed supplement. It is known that L-lysine is produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to its great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to the intrinsic performance characteristics of the microorganisms (e.g., by mutagenesis and selection), to measures concerning fermentation technology (e.g., stirring and oxygen supply), to the composition of the nutrient media (e.g., sugar concentration during fermentation), or to ways in which product is purified or prepared (e.g., ion exchange chromatography or crystallisation.
According to the prior art, there are three different groups of L-lysine-containing product presentations which are produced from fermentation broths. The best known group comprises pulverulent or crystalline product presentations obtained from cleaned-up or pure L-lysine, which is typically present in the form of a salt such as, for example, L-lysine monohydrochloride. Another group of solid product presentations, (see e.g., EP-B-0533039), contain, in addition to L-lysine, the feedstock used during fermentative production and, optionally, the inactivated biomass of the microorganism used. These types of solid products often exhibit clear disadvantages with regard to handling properties and application. For example, the quality of the product may be influenced to a considerable extent by the weather (high air humidity levels may cause caking which impairs the shelf life and dispensing properties of the product). In addition, the processing of dry, pulverulent L-lysine-containing products may lead to undesirable dust development.
The third group of L-lysine-containing products comprises concentrated, aqueous, alkaline solutions. These avoid the above-mentioned problems (EP-B-0534865).
Many different processes are known for producing L-lysine-containing products from L-lysine-containing fermentation broths. There are essentially two different processes currently known for producing solid, pure L-lysine. Lysine may be obtained as a monohydrochloride (Lys-HCl) by crystallisation from the corresponding fermentation broths, after the inactivated biomass has been separated off by suitable methods. Cleaning-up of the filtrate prior to further concentration is performed, in general, by ion exchange chromatography over several stages. Typically, fermentation broth separated from the biomass is acidified, preferably by the addition of hydrochloric acid (HCl) or sulfuric acid (H2SO4), to ease adsorption of the lysine on to ion-exchange resins. One problem with this is that, in addition to L-lysine, various other cations are present in the fermentation broth which are also bound. The adsorbed lysine is then preferably eluted by an ammoniacal solution and the ion-exchange column is regenerated. The lysine solution obtained in this way is then concentrated and lysine-HCl is obtained in crystalline form after neutralisation with hydrochloric acid. In general, a variety of ion-exchange columns connected in sequence are necessary for obtaining a pure product.
Another method enables lysine to be obtained in the form of a crystalline salt after purifying with activated carbon (SU-183581). The lysine-containing fermentation broth is inactivated by standard processes using moist heat and separated from the biomass by filtration. After acidification of the filtrate to pH 5, 4-5% activated carbon is added with constant stirring at 50-55xc2x0 C., in order to separate off undesirable impurities from the filtrate and to prevent discoloration of the crystallizate. In a further filtration step, the activated carbon is separated off and the dissolved sulfate is then precipitated as calcium sulfate by the addition of calcium hydroxide. This is filtered off, the ammonia is removed in a rotary evaporator under vacuum and the solution is concentrated until crystallisation occurs on cooling.
The disadvantage of these two preparation methods lies in the numerous individual stages and the complex cleaning processes using ion-exchange chromatography. The elimination of troublesome salts or the use of different elution media creates additional waste streams, which must either be cleaned up by complex methods or disposed of.
EP-B-0533039 avoids these disadvantages in that all the fermentation feedstock, optionally including the biomass, is concentrated and spray-dried at elevated temperature.
U.S. Pat. No. 5,990,350 describes a process for producing biomass-free L-lysine-containing granules. In this process, the biomass is first isolated from the fermentation broth by ultrafiltration and discarded. The filtrate recovered in this way is then concentrated by extensive evaporation of the water content. The concentrate obtained in this way is finally dried and worked up in a complex manner by fluidised bed spray granulation to produce granules.
A process is also known from EP-B-0534865 for producing aqueous, basic, L-lysine-containing solutions from fermentation broths in which biomass is separated from the fermentation broth and discarded. A pH value of between 9 and 11 is established using a base such as sodium, potassium or ammonium hydroxide. The mineral constituents (inorganic salts) are separated from the broth by crystallisation after concentration and cooling and either used as fertiliser or discarded (land-filled).
The object of the invention is to provide novel, aqueous preparations of L-lysine and its salts suitable as a feed supplement. In addition, it is an object of the invention to provide a production process that is more economical and efficient than processes currently known.
The essential amino acid lysine is widely used as an animal feed supplement. Microorganisms of the genus Corynebacterium are distinguished by their capacity to secrete high concentrations of L-lysine into the corresponding fermentation medium over a relatively short period. The production processes are generally performed as fed batch processes. L-lysine produced by fermentation is currently predominantly worked up to produce a crystalline product, powder or granules.
The present invention provides an animal feed supplement derived from the fermentation broth of Coryneform bacteria, characterised in that it contains
a) L-lysine and/or a salt thereof, preferably at a concentration of 18-35 weight percent (calculated on the basis of lysine base),
b) the biomass formed during fermentation in an amount of from 0 to 100%, preferably from 50 to 100% and more preferably from 90 to 100%, and
c) preferably, greater than 50% of the other dissolved and suspended constituents of said fermentation broth.
The animal feed supplement is in the form of an aqueous composition at a pH of greater than 4 and less than 5.
The animal feed supplement has a lysine content (as lysine base) of 10 wt. % to 35 wt. %, preferably 15 wt. % to 35 wt. %, particularly preferably 18 wt. % to 35 wt. % and very particularly preferably 21 wt. % to 34 wt. %, relative to the total amount of supplement. The pH is less than 7, preferably 2 to 6.5, particularly preferably 2.5 to 6 and very particularly greater than 4 and less than 5.
The total solids content of the supplement amounts to from 10 wt. % to 55 wt. %, preferably 20 wt. to 55 wt., particularly preferably 35 wt. % to 55 wt. % and very particularly preferably 45 wt. % to 54 wt. %. The solids content contains, in addition to lysine preferably the biomass of the producing microorganism, the inorganic and other organic constituents of the fermentation broth and the by-products produced during fermentation, insofar as they have not been separated off by suitable processes such as, for example, filtration.
If the biomass is left completely or partially in the supplement, the latter contains the microorganism protein in a concentration of up to a maximum of 4 wt. %. The supplement contains as inorganic constituents, inter alia, calcium, magnesium, phosphorus in the form of phosphate, and sulfur in the form of sulfate. As organic constituents I contains, inter alia, vitamins (e.g., biotin and thiamine) and sugars (e.g., isomaltose).
The organic by-products formed in small amounts during fermentation include L-amino acids selected from the group comprising L-alanine, L-asparagine, L-glutamine, L-methionine, L-threonine and L-valine. They additionally include organic acids which carry one to three carboxyl groups, such as lactic acid, acetic acid and malic acid. Finally, they also include sugars, such as trehalose. These compounds may be desirable if they improve the valency of the supplement.
The invention further provides a process for producing an aqueous animal feed additive (in the form of a solution or suspension) containing lysine or salts of lysine from a fermentation broth comprising:
(a) producing an L-lysine-containing broth by the fermentation of Coryneform bacteria;
(b) adjusting the pH of the broth to a value of greater than 4 and less than 5.; and
(c) concentrating the broth obtained under vacuum;
wherein the order of steps (b) and (c) is optional.
Mutant coryneform bacteria producing L-lysine are described in detail in the prior art, such as for example in U.S. Pat. No. 4,657,860. For the purpose of L-lysine production, these strains may be cultured continuously or discontinuously using the batch process or the fed batch process or the repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
Examples of suitable fermentation media may be found in patent specifications EP-B-0 532 867, U.S. Pat. No. 5,840,551 and 5,990,350. The culture medium to be used must satisfy the requirements of the particular strains. Carbon sources which may be used include sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose) oils and fats (e.g., soya oil, sunflower oil, peanut oil and coconut oil) fatty acids (e.g., palmitic acid, stearic acid and linoleic acid) alcohols (e.g., glycerol and ethanol) and organic acids (e.g., acetic acid). These substances may be used individually or as a mixture. Nitrogen sources which may be used include organic, nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used include potassium dihydrogen phosphate and dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium should additionally contain salts of metals, such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances, such as amino acids and vitamins, may be used in addition to the above-mentioned substances. Suitable precursors may furthermore be added to the culture medium. The stated feedstock substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid, are used to control the pH of the culture. Foaming may be controlled using antifoaming agents such as fatty acid polyglycol esters. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example, antibiotics. The introduction of oxygen or oxygen-containing gas mixtures such as air into the culture and thorough mixing using suitable stirring systems or the gas stream may be used to maintain aerobic conditions. The temperature of the culture is typically 25xc2x0 C. to 37xc2x0 C. The culture is continued until the maximum quantity of L-lysine has formed. This aim is normally achieved within 10 to 160 hours.
Analysis of L-lysine may be performed by anion exchange chromatography with subsequent ninhydrin derivatisation, as described in Spackman et al. (Analytical Chemistry, 30:1190 (1958)) or it may be performed by reversed phase HPLC, as described in Lindroth et al. (Analytical Chemistry 51:1167-1174 (1979)). The fermentation broths used for the process according to the invention preferably have an L-lysine content greater than 60 g/L (as lysine base) for a content of non-metabolised sugar of less than 5.0 g/L. Out of a total solids content of  greater than 10 wt. %, the biomass preferably accounts for 1 to 4 wt. %. The content of by-products and vitamins from fermentation (amino acids, organic acids) is preferably less than 2 wt.
In the process according to the invention, the biomass present in the fermentation broth is generally inactivated or destroyed at the start, for example, by heat treatment. Inactivation may optionally be dispensed with, however. A pH value of less than 7, preferably 2 to 6.5, particularly preferably 2.5 to 6 and very particularly preferably 2.5 to 5 is then established in the fermentation broth by means of an inorganic acid, such as sulfuric acid, hydrochloric acid or phosphoric acid, or an organic acid, such as citric acid, acetic acid or formic acid, or a mixture of different acids. The biomass is optionally separated off completely or partially by generally known separation or filtration methods before or after acidification. It is not generally necessary to separate off the mineral constituents. The acidic L-lysine-containing solution or suspension is then concentrated using known methods (such as for example a rotary evaporation, film evaporation or falling-film evaporation), preferably under vacuum, until a liquid product is obtained with a lysine content (as lysine base) of 10 wt. % to 35 wt. %, preferably 15 wt. % to 35 wt. %, particularly preferably 20 wt. % to 35 wt. % and very particularly preferably 21 wt. % to 34 wt. %, for a total solids content of 10 wt. % to 55 wt. %, preferably 20 wt. % to 55 wt. %, particularly preferably 35 wt. % to 55 wt. % and very particularly preferably 45 wt. % to 54 wt. %. It is generally the case that the broth is so concentrated that preferably no mineral constituents (inorganic salts) precipitate out of the fermentation broth and the lysine is present in solution. A desired concentration of L-lysine may optionally be established in the product by the addition of an L-lysine-containing substance at any process stage.
The solution/suspension obtained in this way has an acid pH value, is easy to transport, may be readily dispensed, is microbially stable and has a longer shelf life than an alkaline solution. The term xe2x80x9csuspensionxe2x80x9d is used to indicate that the preferably inactivated microorganisms are present in the product in undissolved form.
Acidification may also be performed after or during concentration. The anions acting as sulfate, chloride, phosphate, citrate etc. may be added to the medium in the form of commercially available salts even prior to fermentation.